Hematopoietic stem/progenitor cell (HSPC) transplantation is standard of care for a variety of hematopoietic malignancies and congenital blood diseases. Despite extensive research efforts, the process of stem cell expansion is not fully understood. This proposal aims to understand how dynamic ubiquitination/de-ubiquitination cycles impact hematopoietic stem cell (HSC) expansion through regulation of cytokine signaling. It is based on our previous report that HSC homeostasis is critically controlled by an interaction between JAK2 and the adaptor protein Lnk, which negatively regulates JAK2 activity. Lnk-/- mice harbor a 10-fold expansion in HSCs with superior self-renewal. Lnk deficiency promotes JAK2V617F-induced myeloproliferative disorders (MPDs) in mice. Importantly, acquired Lnk mutations are found in human MPDs, firmly connecting human diseases to these basic studies. Molecular mechanisms by which Lnk regulates JAK2 dependent HSPC expansion, however, remain enigmatic. Here we identified a novel interaction between Lnk and a deubiquitinating enzyme (DUB) complex, BRISC. BRISC (BRCC36 isopeptidase complex), composed of BRCC36, KIAA0157, MERIT40, and BRCC45, specifically cleaves lysine63-linked ubiquitin (K63-Ub) chains - forms of ubiquitin that do not target substrates for degradation, but are involved in signaling transduction. We demonstrated a tyrosine phosphorylation dependent interaction between Lnk and KIAA0157. Notably, endogenous JAK2 is K63-ubiquitinated upon cytokine stimulation, and BRISC depletion heightens cytokine signaling. Furthermore, Merit40-/- mice exhibit increased HSC numbers and enhanced repopulating ability, reminiscent of Lnk-/- mice. Thus, the major goals of this R01 renewal are to define the physiological role of BRISC in HSC function, and relate this to its molecular mechanisms downstream of cytokine signaling. In Aim 1, we will investigate the physiological roles of BRISC in HSPC function, cytokine response, as well as MPD development upon aging. We will also establish the importance of BRISC DUB activity in HSC regulation. Importantly, epistasis experiments will determine genetic interactions between BRISC and the Lnk/JAK2 pathway. In Aim 2, we will investigate mechanisms by which BRISC regulates cytokine signaling. We hypothesize that BRISC regulates HSC self-renewal by modulating the Lnk/JAK2 pathway through K63-ubiquitination/deubiquitnation cycles on JAK2. To test this, we will assess JAK2 K63-Ub levels, JAK2 signaling, and surface expression of JAK2-associated receptors in BRISC null HSPCs. Furthermore, we will test if JAK2 K63-Ub regulates JAK2v617F signaling in MPDs. Additional mechanistic insights will be gained using systematic and unbiased approaches to identify BRISC substrates that potentially affect HSC functions. Hence, our data point to a previously unappreciated role of K63-Ub in HSC biology and cytokine signaling regulation. Our work might provide insights that are beneficial to stem cell transplantation and novel therapies for the treatment of various blood cell disorders with dysregulated HSCs.